Cys2His2 zinc finger proteins can be engineered to recognize a particular DNA sequence, such as a site in the promoter of a particular gene of interest. These proteins, when fused to activation or repression domains, can function as artificial transcription factors. They can be used as tools for the study of gene function in model organisms and potentially as gene therapy reagents for the treatment of disease. However, fundamental questions remain regarding the specificity of these proteins in vivo. This proposal describes the development of dimeric zinc finger proteins that can regulate a single endogenous gene. Dimeric zinc finger proteins should be superior to monomeric zinc finger proteins with regards to sequence specificity and in vivo utility, and they offer a unique system for dissecting the importance of specificity and affinity in in vivo function: Their specificity can be changed by adding or subtracting fingers and their affinity can be varied by increasing or decreasing the strength of the dimerization domain. In Aim #1, we will develop dimeric zinc finger proteins that can regulate a single gene in S. cerevisiae. The in vivo specificity of a series of different dimeric proteins will be characterized, and in conjunction with the analysis of their in vitro specificity and affinity, we will deconvolute the requirements for regulating a single gene in budding yeast. In Aim #2, we will investigate the same question in D. melanogaster. This organism provides unique tools, such as polytene chromosomes, for analyzing the role of specificity and affinity in gene regulation. By defining the increases in specificity and affinity required when moving from yeast to flies to regulate a single endogenous gene, these results should allow us to extrapolate to even more complex organisms such as mice. In Aim #3, we will develop dimeric zinc finger proteins capable of discriminating between two alleles based on single base pair differences. This study should define the limits of single base pair discrimination for zinc finger proteins, and in the future could provide a method for targeting cancer cells by tailoring a therapy specifically to their genotype. Our long-term goal is to develop tools for the regulation of a single gene or allele in a complex genome by understanding the relationship between specificity and affinity and in vivo function.